Azobenzene derivatives as labeling agents and intermediates thereof

ABSTRACT

A compound of the formula I  
                 
 
     wherein R is H or —N═N-2-carboxyphenyl; A is (CH 2 ) n  or —CH═CH—, wherein n is an integer from 0 to 10, or A may also be —CH(COOH)— when R is —N═N-2-carboxyphenyl; and X is a radical selected from the group consisting of: (i) Cl; (ii) COOR 1 , wherein R 1  is p-nitrophenyl or N-succinimidyl; (iii) CONH—NHR 2 , wherein R 2  is H, COO(t-butyl) or COObenzyl; (iv) CONH—[B]—NHR 3 , wherein R 3  is H, COOR 1 , or CO-[B′]-maleimido, wherein R 1  is t-butyl, p-nitrophenyl or N-succinimidyl, and B and B′, the same or different, are (CH 2 ) n  wherein n is an integer from 2 to 10; (v) CONH—[B]—COOR 4 , wherein R 4  is H, C 1 -C 8  alkyl, N-succinimidyl; (vi) CONH—[B]—OH; (vii) CONH—[B]—CONH—NHR 2 , wherein R 2  is H, COO(t-butyl) or COObenzyl; and (viii) NHR 2 , wherein R 2  is H, COO(t-butyl) or COObenzyl, when A is —CH(COOH)— and R is —N═N-2-carboxyphenyl. The 4′-hydroxyazobenzene-2-carboxylic acid (HABA) compounds are novel reagents for labeling, isolation and detection of biological molecules.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a divisional of Ser. No. 09/831,494,filed Aug. 7, 2001, which is the national stage under 35 U.S.C. 371 ofinternational application PCT/IL99/00604, and which the internationalapplication was published under PCT Article 21(2) in the Englishlanguage. There entire contents are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to derivatives of4′-hydroxyazobenzene-2-carboxylic acid (HABA) and to intermediatestherefore, and to their use as novel reagents for labeling, isolationand detection of biological molecules.

BACKGROUND OF THE INVENTION

[0003] There are two major systems currently in use for thenonradioactive labeling of proteins and other biologically activemolecules. These are the avidin-biotin system and the DIG (digoxygenin)system. In both cases, a reactive reagent, containing either the biotinor DIG moiety, is usually used for covalent coupling to a bindingmolecule, e.g. DNA, protein, etc, which in turn will recognize a targetmolecule. Once biotin or DIG is thus incorporated into the experimentalsystem, avidin or DIG-specific antibody (anti-DIG) is appliedsubsequently to serve as a bridge between the target and a probe that isrequired for a desired purpose such as for detection, localization,quantification, isolation, etc.

[0004] Both avidin-biotin and the DIG systems suffer from a series ofdisadvantages. One of the main problems of the avidin-biotin system isthat biotin is a natural vitamin, and as such, it is a normal componentof cells either in the free state or covalently bound to a group ofbiotin-dependent enzymes. Thus, the presence of free biotin willinterfere with the targeting of avidin to a biotinylated molecule,whereas the presence of biotin-dependent enzymes will result in specificbut unwanted binding of avidin. A second disadvantage with theavidin-biotin system is that in some cases a reversible binding betweenavidin and biotin would be desirable, whereas the extremely highaffinity essentially results in an irreversible binding. Another probleminvolves the analysis and processing of a biotinylated preparation.Specifically, since biotin is not chromogenic, it is difficult toquantify the number of biotin moieties per molecule and, more relevant,to determine the percentage of molecules which failed to undergobiotinylation. In this context, it is not easy to separate betweenbiotinylated and non-biotinylated molecules, after the biotinylationstep.

[0005] The DIG:anti-DIG system also suffers from a set of disadvantages.First, it has been noted that DIG reacts nonspecifically with antibodiesfrom serum. Like biotin, the lack of chromophore renders it difficult todetermine the amount of DIG-labeled molecules and to separate them fromthe unlabeled fraction.

[0006] Both systems suffer from another major drawback in that only onetype of protein is available for detection, i.e. avidin or streptavidinfor binding biotin and anti-DIG for DIG.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide new labelingreagents based on derivatives of the azo dye HABA(4′-hydroxyazobenzene-2-carboxylic acid).

[0008] The present invention relates to compounds of the formula I:

[0009] wherein

[0010] R is H or —N═N-2-carboxyphenyl;

[0011] A is (CH₂)_(n) or —CH═CH—, wherein n is an integer from 0 to 10,or

[0012] A may also be —CH(COOH)— when R is —N═N-2-carboxyphenyl; and

[0013] X is a radical selected from the group consisting of:

[0014] (a) Cl;

[0015] (b) COOR₁, wherein R₁ is p-nitrophenyl or N-succinimidyl;

[0016] (c) CONH—NHR₂, wherein R₂ is H, COO(t-butyl) or COObenzyl;

[0017] (d) CONH—[B]—NHR₃, wherein R₃ is H, COOR₁, or CO—[B′]-maleimido,wherein R₁ is t-butyl, p-nitrophenyl or N-succinimidyl, and B and B′,the same or different, are (CH₂)_(n) wherein n is an integer from 2 to10;

[0018] (e) CONH—[B]—COOR₄, wherein B is as defined in (iv) above and R₄is H, C₁-C₈ alkyl, N-succinimidyl;

[0019] (f) CONH—[B]—OH, wherein B is as defined in (iv) above;

[0020] (g) CONH—[B]—CONH—NHR₂, wherein B is as defined in (iv) above andR₂ is H, COO(t-butyl) or COObenzyl; and

[0021] (h) NHR₂, wherein R₂ is H, COO(t-butyl) or COObenzyl, when A is—CH(COOH)— and R is —N═N-2-carboxyphenyl.

[0022] The HABA compounds of formula I, wherein R is—N═N-2-carboxyphenyl, are obtained from the corresponding non-azocompounds wherein R is H.

[0023] In one embodiment, A is (CH₂)_(n) and n is 2 to 4, preferably 2,and the compounds wherein R is H are derivatives of3-(2-hydroxyphenyl)-propionic acid (derivatives (ii) above),-propionamide (derivatives (iv) to (vii) above) and propionic acidhydrazide (derivatives (iii) above). In another embodiments, B ispreferably (CH₂)₅ or (CH₂)₆ and B′ is preferably (CH₂)₃.

[0024] The present invention further relates to conjugates of HABAitself or of a HABA compound of formula I with a carrier (hereinHABAylated compounds), wherein said carrier is a protein or polypeptide,an amino-carrying polymer, a polynucleotide, an oligonucleotide, apolysaccharide, an oligosaccharide or a compound containing a sugarmolecule such as glycocoproteins. Thus the invention encompassesHABAylated cytokines, antibodies, hormones, receptors, DNA, DNA probes,oligonucleotides and other HABAylated molecules.

[0025] In another aspect, the present invention provides anti-HABAantibodies, both polyclonal and monoclonal, which are prepared byimmunization of rabbits and mice, respectively, with a conjugate of HABAand an immunogenic protein, such as for example HABA-KLH.

[0026] The HABA derivatives of formula I can be used as labelingreagents. Labeled molecules are amenable to interaction with eitheravidin or HABA-specific antibodies (anti-HABA) and can be used inseveral applications including, but not being limited to, isolation(affinity chromatography) and detection (immunoassay) of biologicallyactive molecules.

[0027] The invention further provides a method for localization,quantitation and isolation of molecules I in a sample which comprisescontacting the sample with a HABAylated molecule II that recognizesmolecule I, and then reacting with labeled anti-HABA antibodies orlabeled avidin. The HABAylated molecule II such as an HABAylatedantibody, lectin, DNA or RNA, is added to a sample such as a cellpreparation, a DNA or protein blot containing the target molecule II,e.g. the antigen, the carbohydrate, etc, the excess of HABAylated probeis removed after formation of the HABAylated binder II/target molecule Icomplex and then reacted with labeled anti-HABA antibodies or labeledavidin.

[0028] The HABA system has many advantages over both the avidin-biotinand the DIG/anti-DIG systems, as follows:

[0029] HABA can be detected by two unrelated systems: avidin andanti-HABA. Interestingly, the interaction between HABA and anti-HABAgenerates a spectral shift similar to that of the HABA-avidininteraction from 350 to 500 nm. Anti-HABA fails to recognize biotin.

[0030] The HABA moiety is easily detectable and quantifiable, owing toits inherent chromophore and spectral shift upon binding to avidin oranti-HABA.

[0031] It is easy to separate between HABAylated and non-HABAylatedmolecules, due to the reversible interaction with avidin.

[0032] HABAylated molecules can be recognized by avidin and, afteraddition of biotin, the HABAylated molecule is then again available fordetection by anti-HABA antibodies.

BRIEF DESCRIPTION OF THE DRAWING

[0033]FIG. 1 shows the results of ELISA assay using the polyclonalaffinity-purified anti-HABA antibody. Plates were coated with HABAylatedavidin, the desired dilutions of antibodies were applied with biotin (υAntibody purified on column A,  Antibody purified on column B) orwithout biotin (Γ Antibody purified on column A, □ Antibody purified oncolumn B) and the plates were assayed using a secondary antibody-enzymeconjugate.

EXAMPLES

[0034] The invention will now be illustrated by the followingnon-limiting Examples.

List of Compounds

[0035] In the Examples, the following compounds 1-29, which formulas arepresented in Appendix I hereinafter just before the claims, will beidentified by their numbers in bold:

[0036] 0. 4′-hydroxyazobenzene-2-carboxylic acid (HABA)

[0037] 1. 3-(2-hydroxyphenyl)propionic acid

[0038] 2. N-succinimidyl3-(2-hydroxyphenyl)propionate

[0039] 3.N-6-(t-butoxycarbonylamino)hexyl3-(2-hydroxyphenyl)propionamide

[0040] 4. N-6-aminohexyl3-(2-hydroxyphenyl)propionamide

[0041] 5.N-6-(succinimidyloxycarbonylamino)hexyl3-(2-hydroxyphenyl)propionamide

[0042] 6.N-6-(maleimidopropylcarbonylamino)hexyl3-(2-hydroxyphenyl)propionamide

[0043] 7. N-6-(methoxycarbonyl)pentyl3-(2-hydroxyphenyl)propionamide

[0044] 8. N-5-carboxypentyl3-(2-hydroxyphenyl)propionamide

[0045] 9. N-t-butoxycarbonylamino3-(2-hydroxyphenyl)propionamide

[0046] 10. N-6-hydroxyhexyl3-(2-hydroxyphenyl)propionamide

[0047] 11.N-5-(succinimidyloxycarbonyl)pentyl3-(2-hydroxyphenyl)propionamide

[0048] 12.N-5-(t-butoxycarbonylhydrazinocarbonyl)pentyl3-(2-hydroxyphenyl)propionamide

[0049] 13. 3-(2-hydroxyphenyl)propionic acid hydrazide

[0050] 14. N-5-hydrazinocarbonylpentyl3-(2-hydroxyphenyl)propionamide

[0051] 15.3′-(6-t-butoxycarbonylamino)hexylaminocarbonylethyl-4′-hydroxy-azobenzene-2-carboxylicacid

[0052] 16.3′-(6-aminohexylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0053] 17.3′-(6-(succinimidyloxycarbonylamino)hexylaminocarbonylethyl)-4′-hydroxy-azo-benzene-2-carboxylicacid

[0054] 18.3′-(6-(maleimidopropylcarbonylamino)hexylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0055] 19.3′-(1′-carboxy-t-butoxycarbonylaminomethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0056] 20.3′-(5-carboxypentylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0057] 21.3′-(5-succinimidyloxycarbonylpentylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0058] 22.3′-(5-t-butyloxycarbonylhydrazinocarbonylpentylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0059] 23.3′-(t-butyloxycarbonylhydrazinocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0060] 24.3′-(6-hydroxyhexylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0061] 25.3′-(5-(hydrazinocarbonyl)pentylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0062] 26.3′-(hydrazinocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylic acid

[0063] 27. 3′-(carboxyethyl)-4′-hydroxy-azobenzene-2-carboxylic acid

[0064] 28.3′-(succinimidyloxycarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylicacid

[0065] 29.3′-(1-carboxy-1-amino-methyl)-4′-hydroxy-azobenzene-2-carboxylic acid

[0066] ABBREVIATIONS: BOC: t-butoxycarbonyl; BSA: bovine serum albumin;DCC: N,N′-dicyclohexylcarbodiimide; DMAP: dimethylaminopyridine; DMF:N,N′-dimethyl formamide; DSC: disuccinimidylcarbonate; HABA:4′-hydroxyazobenzene-2-carboxylic acid; KLH: Keyhole Lympet Hemocyanin;NHS: N-hydroxysuccinimide; Su: succinimidyl; TEA: triethylamine; TSTU:tetramethyluronium tetrafluoroborate.

[0067] REAGENTS and INSTRUMENTATION: DCC and TEA were obtained fromMerck (Darmstadt, Germany). N-BOC-1,6-diaminohexane,N-BOC-1,6-diaminoetane, ε-amino-caproic acid and 6-aminohexanol, wereobtained from FLUKA Chemie (Buchs, Switzerland). DSC was purchased fromCalbiochem/NovaBiochem (La Jolla, Calif., USA).3-(2-hydroxy-phenyl)propionic acid, 2-hydroxycinnamic acid, anthranilicacid and anhydrous hydrochloric acid solution in dioxane were purchasedfrom Aldrich (Milwaukee, Wis., USA). KLH was gently provided by Biomakor(Israel). Avidin was provided by STC Laboratories (Winnipeg, Canada).β-Maleimidopropionic acid N-hydroxysuccinimidyl ester,N-hydroxy-succinimide, biotin, DL-O-tyrosine, HABA, BSA, and all theother chemicals were obtained from Sigma Chemicals (S. Louis, Mo., USA).Sepharose CL-4B was purchased from Pharmacia Biotech AB (Uppsala,Sweden). BCA protein assay reagent was obtained from Pierce (Rockford,Ill., USA). Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)and anti-rabbit IgG were obtained from Jackson ImmunoResearchLaboratories (West Grove, Pa., USA).

[0068] UV spectra were recorded with a Milton Roy Spectronic UV-VisSpectrophotometer, mod. 1201. Methods of synthesis of3-(2-hydroxyphenyl)propionic acid derivatives (Compounds 2-14) and ofthe HABA derivatives (Compounds 15-29).

[0069] The methods of synthesis of compounds 2-29 are depicted inSchemes 1-8 hereinafter just before the Claims, and are summarized asfollows:

[0070] (i) The HABA derivatives are prepared by diazotization of the3-(2-hydroxyphenyl)-propionic acid derivatives with anthranilic acid andsodium nitrite according to standard procedures.

[0071] (ii) Compounds containing a Su-O—CO-[A]-or a Su-O—CO—[B]— groupare prepared from the corresponding free carboxylic acids either byactivation with NHS or with TSTU, according to procedures a and b,respectively, in Scheme 1. Thus, for example, Compounds 2, 11, 21, 28,are obtained by reaction of Compounds 1, 8, 20, 27, respectively, eitherwith (a) NHS in the presence of DCC, or (b) with TSTU and DMAP.

[0072] (iii) Compounds containing a BOC—NH—[B]—NH— group such as forexample, Compound 3, are prepared by reaction of the correspondingSu-O—CO-[A]-compounds obtained as in (ii) above, for example Compound 2,with a BOC—NH—[B]—NH₂ compound as shown in Scheme 2A. The HABAderivative such as for example Compound 15, can then be prepared bydiazotization, as depicted in Scheme 2B.

[0073] (iv) Compounds containing a H₂N—[B]—NH— group such as forexample, Compounds 4 and 16, are prepared by removal of the protectiveBOC group from the corresponding BOC—NH—[B]—NH— compounds obtained as in(iii) above, for example from Compounds 3 and 15, respectively (Scheme2B).

[0074] (v) Compounds containing a Su-O—CO—NH—[B]—NH— group such as forexample, Compounds 5 and 17, are prepared by reaction of thecorresponding H₂N—[B]—NH— compounds obtained as in (iv) above, forexample Compounds 4 and 16, respectively, with DSC in the presence ofTEA (Scheme 2C).

[0075] (vi) Compounds containing a maleimido-[B′]—CO—NH—[B]—NH— groupsuch as for example, Compounds 6 and 18, are prepared by reaction of thecorresponding H₂N—[B]—NH— compounds obtained as in (iv) above, forexample Compound 4 and 16, with N-Su 3-maleimidopropionate in thepresence of TEA, according to Scheme 3.

[0076] (vii) Compounds containing an alkyl-O—CO—[B]—NH— group such asfor example, Compound 7, are prepared by reaction of the correspondingfree carboxylic acids, for example Compound 1, with an ω-aminoalkanoicacid ester alkyl-O—CO—[B]—NH₂, in the presence of DCC/TEA.

[0077] (viii) Compounds containing a HOOC—[B]—NH— group such as forexample Compound 8, are prepared by hydrolysis of the correspondingesters alkyl-O—CO—[B]—NH— obtained as in (vii), for example fromCompound 7.

[0078] (ix) Compounds containing a BOC—NH—NH—CO-[A]-or BOC—NH—NH—CO—[B]—group such as for example, Compounds 9 and 12, respectively, areprepared by reaction of the corresponding Su-O—CO-[A]-or Su-O—CO—[B]—compound, for example Compounds 2 and 11, respectively, withBOC-hydrazine. The HABA derivatives such as for example Compounds 22 and23, can then be prepared by diazotization from Compounds 12 and 9,respectively.

[0079] (x) Compounds containing a HO—[B]—NH—CO-[A]— group such as forexample, Compound 10, are prepared by reaction of the correspondingSu-O—CO-[A]-compound, for example Compound 2, with a HO—[B]—NH₂compound. The HABA derivative such as for example Compound 24, can thenbe prepared by diazotization from Compound 10.

[0080] (xi) Compounds containing a H₂N—NH—CO-[A]- or H₂N—NH—CO—[B]—NH—group such as for example, Compounds 13 and 14, respectively, and theHABA derivatives 26 and 25, respectively, are prepared by removal of theprotective BOC group from the corresponding BOC—NH—NH—CO-[A]- orBOC—NH—NH—[B]—NH— compounds obtained as in (ix) above, for exampleCompounds 9 and 12, and Compounds 22 and 23, respectively.

Example 1 Synthesis of Derivatives of 3-(2-hydroxyphenyl)propionic acid1.1 Synthesis of Compound 2

[0081] According to method (ii) above, to a cooled solution of Compound1 (0.997 g, 6 mmoles) in CH₂Cl₂ (21 ml), NHS (0.828 g, 7.2 mmoles) andDCC (1.485 g, 7.2 mmoles) were added. After 3.5 h, the solutioncontaining Compound 2 was filtered and directly used for the nextsynthetic step, without any further purification.

1.2 Synthesis of Compound 3

[0082] According to method (iii) above, N1-BOC-1,6-diaminohexane (1.52g, 6 mmoles) was added, while stirring, to the dichloromethane solutionof Compound 2, followed by 835 ml (6 mmoles) of TEA. The reaction wasstirred overnight at room temperature, filtered and evaporated todryness. The product was redissolved in ethyl acetate and the organicsolution was washed (with diluted NaHCO₃, diluted citric acid andwater), dried over Na₂SO₄ and evaporated to dryness. Diethyl ether (30ml) was added to the resulting oil, the precipitated impurities wereremoved by filtration, and the solution containing Compound 3 wasevaporated to dryness and used further.

1.3 Synthesis of Compound 4

[0083] According to method (iv) above, Compound 3 (1 g) dissolved indioxane (40 ml) was treated with HCl-saturated dioxane. After one hour,the precipitate containing Compound 4 was filtered, washed with diethylether and dried.

1.4 Synthesis of Compound 5

[0084] According to method (v) above, a solution of Compound 4 (0.5mmoles) in DMF (1.6 ml) was slowly added in portions (8×200 ml), whilestirring, to a DSC solution in CH₃CN (256 mg, 1 mmole, in 10 ml). Aftereach addition, 2 equivalents of TEA (with respect to compound 4) werealso added, and the pH monitored continuously and maintained below 4.0.Five minutes after the last addition of Compound 4, 5 ml of 1N HCl wereadded. The product Compound 5 crystallized as a fine powder, wasisolated by filtration, washed with diluted HCl and dried.

1.5 Synthesis of Compound 6

[0085] According to method (vi) above, Compound 4 dissolved in DMF (0.05mmoles in 500 ml) was added to a solution of N-succinimidyl 3-maleimidopropionate (26.6 mg, 0.1 mmole) in 1.5 ml CH₃CN/DMF 3:1, followed by 0.1mmoles TEA. After 2 hours, 3 ml of H₂O were added and, afteracidification with 1N HCl, the product Compound 6 crystallized.

1.6 Synthesis of Compound 7

[0086] According to method (vii) above, to a solution of Compound 1(0.997 g, 6 mmol) in CH₂Cl₂ (25 ml) were added ε-aminocaproic acidmethyl ester (1.74 g, 12 mmol), an equimolar amount of TEA (1.6 ml, 12mmol) and DCC (1.36 g, 6.6 mmol). The reaction was carried out for 4hours in an ice bath. The solution was washed thoroughly with water, HCl(0.05 M), water, bicarbonate (0.1 M) and again with water. The CH₂Cl₂fraction was dried over sodium sulfate and the pure product Compound 7was obtained by precipitation with absolute diethylether.

1.7 Synthesis of Compound 8

[0087] According to method (viii) above, Compound 7 (330 mg, 1.08 mmol)was dissolved in methanol and 5.4 ml 0.5 M NaOH were added thereto.After 1 hour, the reaction mixture was brought to pH 2 with HCl and themethanol removed by evaporation. The oily mixture was dissolved in hotethyl acetate and the pure product Compound 8 crystallized upon coolingdown.

1.8 Synthesis of Compounds 9 and 12

[0088] According to method (ix) above, Compounds 9 and 12 weresynthesized from Compounds 2 and 11, respectively, by reaction withBOC-hydrazine, according to the procedure described in section 1.2 abovefor the preparation of compound 3.

1.9 Synthesis of Compound 11

[0089] According to method (ii) above, Compound 11 was obtained fromcompound 8, using the same procedure described in section 1.1 above forthe preparation of compound 2.

1.10 Synthesis of Compound 10

[0090] According to method (x) above, Compound 10 was obtained fromcompound 2 and 6-aminohexanol, using the same procedure described insection 1.2 above for the preparation of compound 3.

1.11 Synthesis of Compounds 13 and 14

[0091] According to method (xi) above, Compounds 13 and 14 were obtainedby removing the BOC protective group from Compounds 9 and 12,respectively, using the same procedure described in section 1.3 abovefor the preparation of Compound 4.

Example 2 Synthesis of HABA-Derivatives 2.1 Synthesis of Compound 15(HABA-C₂—CONHC₆—NH—BOC)

[0092] According to method (i) above, to cooled anthranilic acid (0.750g, 5.45 mmoles) and NaNO₂ (0.377 g, 5.45 mmoles) dissolved in water (15ml), 1.5 ml of concentrated HCl were added. After 10 min, the solutionwas dropwise added to Compound 3 (1.62 g, 5.45 mmoles) dissolved in amixture of methanol/0.5M KOH :1/1 (15 ml). The pH was controlled andadjusted to 8.0 using HCl and KOH. After 20 min, methanol was removed byevaporation, the solution was acidified to pH 3-4 with diluted citricacid, and the solid product was extracted with ethyl acetate. Theorganic solution was washed with water, dried over Na₂SO₄ and evaporatedto dryness, thus obtaining Compound 15.

2.2 Synthesis of Compound 16 (HABA-C₂CONH—C₆—NH₂×HCl)

[0093] According to method (iv) above, a solution of Compound 15 indioxane was dried, filtered, and HCl saturated dioxane was added. After1 hour, the product Compound 16 precipitated as the hydrochloride salt,was isolated by filtration, washed with diethyl ether and dried.

2.3 Synthesis of Compound 17 (HABA-C₂—CONH—C₆—NH—OSu)

[0094] According to method (v) above, a solution of Compound 16 (35.8mg, 0.08 mmoles) in DMF (0.24 ml) was slowly added into portions (8×30μl), while stirring, to a solution of DSC (41 mg, 0.16 mmoles) in CH₃CN(1.6 ml). After each addition, 2 equivalents of TEA (with respect toCompound 16) were also added, and the pH monitored continuosly andmaintained below 4.0. Five minutes after the last addition of theHABA-derivative 16, 2 ml of 1N HCl were added. The product Compound 17crystallized as a fine powder. It was isolated by filtration, washedwith diluted HCl and dried.

2.4 Synthesis of Compound 18 (HABA-C₂—CONH—C₆—NHCO—C₃—N- Maleimide)

[0095] According to method (vi) above, N-succinimidyl3-maleimidopropionate (31.5 mg) was dissolved in 2 ml of CH₃CN/DMF 3/1.Compound 16 was dissolved in DMF (25 mg in 500 ml) and then added to themaleimido derivative solution, followed by 21 ml of TEA. After 3 hours,5 ml of H₂O were added and, after acidification with 1N HCl, the productCompound 18 crystallized.

2.5 Synthesis of Compounds 19, 20, 22, 23, 24, 27

[0096] Compounds 19, 20, 22, 23, 24, 27 were synthesized from theCompounds N—BOC-DL-o-tyrosine, 8, 12, 9, 10 and 1, respectively,following the same procedure described in section 2.1 above for Compound15.

2.6 Synthesis of Compounds 25 and 26

[0097] According to method (xi) above, Compounds 25 and 26 were obtainedfrom Compounds 23 and 22, respectively, by acid hydrolysis, followingthe same procedure described in section 2.2 above for Compound 16.

2.7 Synthesis of Compound 21

[0098] According to method (ii) above, Compound 21 was prepared fromCompound 20 by two different procedures:

a. Activation with NHS (Scheme 1a)

[0099] The synthesis was carried out using the same procedure describedin Example 1.1 above for Compound 2. Compound 20, DCC/CH₂Cl₂ and NHSwere used in equimolar concentrations to avoid activation of thecarboxyl group at the second phenyl ring. The urea derivative wasremoved by filtration, and Compound 21 was washed with water and dried.

b. Activation with TSTU (Scheme 1b) (Bannwarth 1991)

[0100] TSTU (70.4 mg, 0.24 mmol) and DMAP (57 mg, 0.48 mmol) were addedto Compound 20 (100 mg, 0.24 mmol) dissolved in a mixture ofDMF/dioxane/water (1/1/0.5). After complete conversion (30 min),Compound 21 (purity 96%) was lyophylized and further purified by HPLC.

2.8 Synthesis of Compound 28

[0101] According to method (ii) above, Compound 28 was prepared fromCompound 27 by two different procedures:

a. Activation with NHS (Scheme 1a)

[0102] The synthesis was carried out as described in section 2.7.a abovefor Compound 21.

b. Activation with TSTU (Scheme 1b) (Bannwarth 1991).

[0103] TSTU (100 mg, 0.32 mmol) and DMAP (80 mg, 0.64 mmol) were addedto Compound 27 (100 mg, 0.32 mmol) dissolved in a mixture ofDMF/dioxane/water (1/1/0.5). Complete conversion occurred after 30minutes. Compound 28 (purity 95%) was lyophylized and further purifiedby HPLC.

2.9 Synthesis of Compound 29

[0104] Compound 29 was obtained from Compound 19 upon acidic cleavage ofthe BOC group, following the same procedure described in section 2.2 forCompound 16.

2.10 Synthesis of further HABA-Derivatives

[0105] HABA-derivatives carrying different spacers A are obtained by thesame procedures described above but using different derivatives asstarting materials instead of 3-(2-hydroxyphenyl)propionic acid(Compound 2). For example, compounds wherein A is CH═CH are obtainedusing 2-hydroxycinnamic acid derivatives as starting materials; when Ais (CH₂)_(n) and n is zero, from salicylic acid derivatives; when n=1,from 2-hydroxyphenylacetic acid derivatives; when n=3, from4-(2-hydroxyphenyl)butyric acid derivatives; and when n>3, fromX-(2-hydroxyphenyl) acid derivatives (X=n+1) with the desired (n) chainlength. These acids can be obtained by malonic synthesis as described inBeil. III, 10, 586-587 for the butyric acid derivatives.

Example 3 Labeling of Proteins and Other Compounds with HABA 3.1Chemistry of Binding

[0106] The 2-hydroxyphenyl- or HABA-derivatives that have been activatedas N-Su carbamates (—NH—CO—O-Su) or as N-Su esters (—CO—O-Su) react withprimary amino groups at the level of any protein, peptide oramino-carrying polymer (e.g. Sepharose-diaminohexane, etc.) surface. Inproteins or peptides, the binding occurs through the lysyl-ε-NH₂ or theα-NH₂ of the first amino acid of the chain. The coupling reactions aredepicted in Schemes 4 and 5 and lead to urea and carbamide types ofbond, respectively.

[0107] The 2-hydroxyphenyl- or HABA-derivatives functionalized with aterminal maleimido group are thiol-specific reagents. In proteins orpeptides, the binding occurs at the level of cysteine SH functions. Thereaction is depicted in Scheme 6.

[0108] The 2-hydroxyphenyl- or HABA-derivatives functionalized with aterminal hydrazido group react with any aldehyde residue. In proteins,glycoproteins and carbohydrates in general, the reaction occurs, afterperiodate or enzymatic oxidation, at the level of the sugar aldehydicresidues. The reaction is depicted in Scheme 7.

3.2 Protein Modification with HABA Reagents (HABAylated Proteins) 3.2.aSuccinimidyl Esters (Compounds 21 and 28)

[0109] In a general coupling reaction according to Scheme 5, a freshlyprepared solution of a HABA-N-Su ester (—CO—O-Su, 2.5 ml) in EtOH:PBS1:3 (1-50 mg/ml) is added, while stirring, to a solution of a proteinP—NH₂ (2-20 mg) in 1 ml of phosphate buffer, pH 7.4 containing 0.5MNaCl. The protein P—NH₂ is, for example, a protein to be used fordetection as a binder, e.g. an antibody. The molar ratio between theHABA-N-Su ester derivative and the protein is between 4 and 100depending on the starting protein concentration, the reactivity of thespecific protein and the desired degree of modification. The reaction iscarried out at room temperature or lower for several hours. Examples ofHABAylation procedures using succinimidyl esters 21 and 28 are shown andsummarized in Table 1 for several proteins. TABLE 1 HABAylation ofproteins - Reaction conditions and products obtained. HABA/ proteinProtein HABA/ Obtained conc. in protein with residual coupling used andComp. 21 & activity Protein (mg/ml) react. Time 28 % BSA 10  16 (12 h)2/2 — 10  40 (12 h) 4/5 — KLH 1 300 (12 h) 282/400 — 1 300 (½ h.)118/256 — Ribonuclease 10  4 (12 h)   1/0.5 — 10  30 (2 h)   3/2.5 —γ-Globulin 1 345 (2 h)  7/11 — 1 100 (2 h)   5/8.5 — Lysozyme 10  4 (12h) 1/1 — 10  30 (2 h) 2.5/2   — α-Lactalbumin 10  40 (1 h) 2/1 — Sheepanti- 2.3 150 (12 h) 16/12 100 rabbit IgG 2.3 150 (½ h.) 9/7 100 MouseIgG 17 200 (½ h.) 7/5 100 Anti-L-amino 2 100 (½ h.) 19/14  98 acids 2 50 (½ h.) 11/6  100

3.2.b Succinimidyl Carbamates (Compound 17)

[0110] In a general soupling reaction according to Scheme 4, 10-50 ml ofa concentrated solution of a HABA-Su carbamate (—NH—CO—O—Su) in DMF(5-30 mg/ml) are added, while stirring, to a solution of a protein P—NH₂in aqueous buffer at pH 8.0-8.5 (2-10 mg/ml). The protein P—NH₂ is, forexample, a protein to be used for detection as a binder, e.g. anantibody. The molar ratio between the HABA-Su carbamate derivative andthe protein is between 50 and 400, depending on the starting proteinconcentration, the reactivity of the specific protein and the desireddegree of modification. The reaction is carried out at room temperatureor lower for 1-2 hours. Examples of HABAylation procedures, reactionconditions and products using succinimidyl carbamates (Compound 17) areshown and summarized Table 2 for several proteins. TABLE 2 HABAylationof proteins - Reaction conditions and products obtained HABA/ ProteinHABA/ protein conc. in protein obtained residual coupling used and withactivity protein (mg/ml) react. time Comp. 17 % Goat-anti- 3.19  50 (1h.) 6 100 Mouse IgG 3.19 100 (1 h.) 6.5 100 3.19 200 (1 h.) 8  90 3.19400 (1 h.) 11  85 Ovoalbumin 2.5  20 (½ h.) 0.5 — 9.9  20 (½ h.) 2 — KLH10 300 (½ h.) 51 — 50  60 (½ h.) 20 — 60  15 (½ h.) 1.5 — BSA 5 150 (24h) 20 — 5 150 (24 h) 16 — HRP 2 300 (2 h) 3 100 7.7  35 (3 h) 0.5 100

3.2.c Maleimido Derivatives (Compound 18)

[0111] According to Scheme 6, HABAylation of cysteine residues inproteins and peptides P—SH is carried out by a coupling reaction withmaleimido derivatives, either using an excess of peptide, thus obtaininga mixture of HABAylated and non-HABAylated products, or using a largeexcess of the HABA reagent, in which case complete HABAylation isobtained.

[0112] For HABAylation of glutathione, 50 ml of a HABA-maleimidesolution in methanol (20 mg/ml) were mixed with 2 mg of GSH, previouslydissolved in 2 ml of PBS. After 5 min, total disappearance of themaleimido reagent can be verified by TLC (CHCl₃/MeOH 20%).

[0113] For HABAylation of the single cystein residue (Cys 62) ofCellulose Binding Domain (CBD) from Clostridium Thermocellum YS, asolution of the HABA-maleimide reagent in methanol is added to asolution of CBD in PBS in a 2 fold excess.

3.2.d HABA-Hydrazido Derivatives (Compounds 25 and 26)

[0114] According to Scheme 7, aldehyde residues of glycoproteins andpolysaccharides are modified using protocols described elsewhere(Wilchek and Bayer, 1987).

3.2.e Protein Modification with 3-(2-hydroxyphenyl)propionic acidderivatives (Compounds 2, 5, 6, 11, 13, 14)

[0115] Proteins can be modified with 3-(2-hydroxyphenyl)propionic acidderivatives by similar procedures as described in 3.2.a -3.2.d aboveaccording to Schemes 5, 6 and 7 (R=H).

3.3 Purification of the HABAylated Proteins

[0116] The products according to 3.2.a -3.2.e above are purified fromlow molecular weight molecules such as excess of reagent and NHS in thecase of succinimidyl reagents, by overnight dialysis at 4° C. or by gelfiltration on a G25 column. In this case, 3 mg of protein mixture areapplied to a column of diameter 0.5 cm×h: 20 cm, and PBS or any desiredbuffer is used as eluant.

[0117] If the final reaction mixture of any HABAylation contains bothnon-HABAylated and HABAylated products, the two fractions can beseparated by affinity chromatography using a Sepharose-avidin columnthat retains the HABAylated molecules. A highly protein concentratedSepharose-avidin gel (5 mg avidin/g wet gel), prepared by cyanogenbromide or other activation method as described by Wilchek et al, 1984,is used for this purpose. PBS is used as washing buffer to elute theunmodified fraction from the gel. The retained protein or peptide isthen eluted with HABA, biotin, salts or 50 mM, pH 10.5 TEA, depending onthe amount of HABAylation. When the basic elution is carried out,fractions are immediately neutralized using 0.2 M acetic acid. If biotinsolution is used to elute the HABAylated molecule, the avidin columnwill have to be treated under conditions which dissociate avidin andbiotin.

3.4 Characterization of the HABAylated Proteins 3.4.a ProteinConcentration and Degree of HABAylation

[0118] Protein concentration after coupling and purification isestimated by BCA (bicinchoninic acid) protein assay (Pierce) or by usingthe UV absorption values and keeping into account the influence of theHABA residues at 280 nm (ε_(280 nm) for compound 16=3,300). The degreeof modification is calculated, after purification from side-products, onthe basis of UV absorption at 356 nm in PBS, at 415 nm in basicenvironment, or at 504 nm after addition of excess of avidin in PBS.TABLE 3 Spectroscopic characteristics of HABA (Compound 0) in differentconditions. Conditions λmax (nm) ε [cm⁻¹M⁻¹] PBS 350 20,500 + affinitypurified anti- 482 50,000 HABA in PBS, (1:1) + Avidin in PBS, (20:1) 50035,500

[0119] 0In the case of proteins containing chromophores, e.g. KLH, whichhas a chromophore with a maximum of absorption at 344 nm, absorption ofthe native protein has to be taken into account in all calculations.

3.4.b Interaction of Avidin and Anti-HABA Antibodies withHABA-Derivatives

[0120] Interaction of the HABAylated proteins with avidin or withanti-HABA antibodies is verified by following the characteristic shiftof the absorption of the HABA chromophore. Table 4 depicts thespectroscopic characteristics in the spectra range of theHABA-derivatives as free molecules (not bound to proteins) uponinteraction with avidin or specific affinity purified antibodies. TABLE4 Molar Extinction Coefficients for HABA-derivatives ε [cm⁻¹M⁻¹] atCompound Buffer/conditions λmax (nm) λmax 16 PBS 356 12,900 16 0.1 MAcOH 356 12,500 16 0.1 M NaOH 420 15,000 16 PBS/AVIDIN 504 30,000 16PBS/anti-HABA 482 50,000 17 0.1 M AcOH 356 12,200 27 [A] = (CH═CH) PBS356 15,500 27 [A] = (CH═CH) PBS/AVIDIN 520 33,000 27 [A] = (CH═CH)PBS/anti-HABA 505 50,000

Example 4 HABA-Sepharose with High Degree of Functionalization

[0121] A high degree of Sepharose HABAylation can be obtained by usingthe synthetic approach described in Scheme 8. The gels obtained areintensely colored in dark red and have a high affinity and capacity ofavidin (˜20 mg/g wet gel). On the other hand, proteins such as thosecontained in the egg yolk, e.g. ovalbumin, ovomucoid, conalbumin,riboflavin-binding protein and lysozyme, are not retained by the gels.HABA-Sepharose can therefore be used to extract avidin from any solutionor for its purification from egg yolk.

[0122] The HABA-Sepharose column can also be applied for affinitypurification of polyclonal anti-HABA antibodies from serum.

4.1 Synthesis of HABAylated-Sepharose Gel

[0123] According to Scheme 8, Sepharose CL-4B hydroxyl functions arefirst activated as p-NO₂ phenyl carbonates as described in Wilchek etal, 1984, and the active gel is then coupled to o-tyrosine or to a3-(2-hydroxyphenyl)alkanoic acid derivative carrying a spacer arm with aprimary amine as the terminal group such as Compound 4, or similarcompounds with different spacers A and/or B. The HABA function is thenobtained by diazotization of the phenyl residues directly on theSepharose support (Vetter 1994).

4.1.a Synthesis of Sepharose-Tyrosine and Sepharose-Compound 4

[0124] A solution of o-tyrosine or Compound 4 in aqueous buffer (35 mMin bicarbonate or borate, pH 8.5) is added to p-NO₂-phenylcarbonate-activated Sepharose carrying 50-100 mmoles active groups/g wetgel.

[0125] The reaction is carried out on 3-5 g gel in a total volume of12-15 ml, using a 3:1 molar ratio between the primary amine (o-tyrosineor Compound 4) and the activated groups of the gel. The suspension isgently stirred for 150 minutes at room temperature. The gel is thenwashed with H₂O, MeOH, EtOAc and then, MeOH and water again. Unreactedactive groups in the gel are hydrolyzed by 5 minutes exposure to 0.2MNaOH. The gel is washed again and resuspended in 0.2M KOH (3 g/5 ml) forthe final diazotization step.

4.1.b Synthesis of HABAylated-Sepharose: Diazotization Reaction

[0126] Anthranilic acid and NaNO₂ are dissolved in H₂O (156 mmoles/mlfor both) and concentrated HCl (100 pl/ml of water) is added aftercooling in a ice bath. The solution is stirred for 5 minutes and thenadded dropwise to the gel suspended in 0.2M KOH (3 g/5 ml). The reactionis gently stirred for 15 minutes under temperature control. The pH ismonitored constantly and adjusted to 8.0-8.5 using diluted KOH. A molarratio of 1:1 between anthranilic acid and the phenyl residues in the gelis used, assuming that a complete conversion of the activatedp-NO₂-phenyl groups occurred in the previous step of the synthesis. Thegel is then washed using the procedure as in the previous step and it isfinally suspended in PBS.

4.2 Avidin Purification using HABAylated-Sepharose

[0127]FIG. 1 shows the elution profiles of avidin fromHABAylated-Sepharose obtained according to the method described in 4.1.babove using Compound 4. The gel (100 mg) was incubated for 20 min atroom temperature with an avidin solution in PBS (2 mg/ml) and thenwashed with different eluants. Final elution of avidin was carried outwith either 0.3M TEA, pH 11.5 or with a biotin solution in NaHCO₃(1mg/ml). When the TEA buffer is used, the eluted fractions areimmediately neutralized using IM acetic acid.

Example 5 Anti-HABA Antibodies: Production and Characterization 5.1Preparation of Immunogenic HABAylated Proteins

[0128] KLH, BSA, and goat-affinity purified anti-Mouse IgGs wereHABAylated using the succinmidyl carbamate reagent (Compound 17) as thecoupling agent by the procedure described in 3.2.b above. Briefly, thecoupling agent dissolved in DMF was added to a solution of the proteinin 0.1M NaHCO₃ (2-10 mg/ml). After 2 hours, the excess of HABA reagentwas removed by gel filtration on a G25 column. The degree of couplingcould be estimated from the UV spectra of the conjugates, consideringthe ε₃₅₆ of 12,900 for the HABA-derivative in PBS and measuring theprotein concentration by BCA protein assay.

5.2 Preparation of Anti-HABA Polyclonal Antibodies 5.2.a RabbitImmunization for Anti-HABA Production

[0129] Rabbits (12 weeks) were immunized by intradermal injection of 0.5mg of HABA-KLH (carrying 50 molecules of HABA/protein) emulsified incomplete Freund's adjuvant. Boosts were administered after 4 weeks byinjecting 0.5 mg of HABA-protein in incomplete Freund's adjuvant. Bloodwas collected from the ear vein two weeks after boosting and serum wasisolated by centrifugation and preserved at −20° C. Preserum wascollected before immunization and used as a control.

5.2.b Affinity Purification of Anti-HABA Polyclonal Antibodies

[0130] HABA-Sepharose coupled with Compound 4 with diaminohexane asspacer arm was prepared from Sepharose CL-4B as described above in4.1.a. The gel was pre-treated with 0.1M TEA pH 11.5 before any furtheruse and re-equilibrated with PBS. Rabbit antisera diluted 1:1 with PBSwere incubated with the gel for 4 hours at 4° C. Total removal ofanti-HABA antibodies from supernatant was verified by dot blot onnitrocellulose paper, using BSA-HABA for dotting. In order to obtain anefficient retention of anti-HABA antibodies, a ratio of 1:1 w/w betweenserum and gel was used. The gel was then washed extensively with 0.05 MTris HCl, 0.5M NaCl, pH 7.5, and bound antibodies were eluted by basictreatment using 0.1M TEA, pH 11.5. The eluting fractions wereimmediately neutralized and dialyzed against PBS. Alternatively, thePierce gentle Ab/Ag elution buffer was used. No significant differencebetween the antibodies eluted with these two eluants was observed.

5.2.c Characterization of Anti-Sera and Affinity Purified Anti-HABAPolyclonal Antibodies

[0131] Both sera and affinity purified antibodies are analyzed forantigen recognition by ELISA assay according to the following protocol:96-well microtiter plates (Nunc F96 Maxisorp) are coated by overnightincubation at 4° C. with 50 μl/well of a BSA-HABA solution 10 μg/ml in0.05 M NaHCO₃, pH 9.3. Plates are washed three times with PBS/Tween0.05% (PBS/T) and blocked by adding 200 μl of PBS/T containing 3% BSA.After 2 hours incubation at 37° C., plates are washed three times withPBS/T.

[0132] Serial dilutions of anti-sera or affinity purified antibodies (50μl) are incubated for 2 h at 37° C. The plates washed 3 times with PBS/Tare incubated for 2 hours at 37° C. with 50 μl/well of a solutioncontaining HRP-conjugated anti-rabbit antibodies (diluted 1:2500). Afterextensive washing with PBS/T, 100 μl of ABTS solution are added and theOD. at 405 nm is measured after 30 minutes (Engvall 1980). The purity ofthe affinity purified anti-HABA polyclonal antibodies is verified by SDSgel electrophoresis.

5.3 Affinity Purification of Anti-HABA Polyclonal Antibodies

[0133] Two different Sepharose gels (A and B) were prepared for theisolation of anti-HABA antibodies specific to different epitopes in theHABA molecule. A schematic representation of the two gels is describedin Scheme 9.

[0134] GEL A is a highly functionalized HABAylated-Sepharose having thecorrect HABA moiety (4′-hydroxy-azophenyl-2-carboxylic acid linked atposition 3′) connected with a spacer arm in a similar way as in theHABAylated KLH.

[0135] GEL B is a highly functionalized gel having a HABA moiety with aslightly different structure (2′-hydroxy-azophenyl-2-carboxylic acidlinked at position 5′ via a spacer arm) obtained by diazotization oftyramine-Sepharose with anthranilic acid.

[0136] These two gels allow isolation of anti-HABA antibodies withdifferent characteristics: GEL A is able to isolate anti-HABA antibodiesthat recognize the whole HABA molecule, while GEL B allows the isolationof antibodies that are specific for the azophenyl-2-carboxylic acidmoiety of the HABA core.

5.3. a Preparation of GEL A

[0137] Sepharose CL-4B hydroxyl functions were first activated asp-nitrophenyl carbonates (Wilchek et al, 1984) and the active gel wasthen coupled to a 2-hydroxyphenyl derivative carrying a spacer arm witha primary amine as the terminal group. Different spacers can beintroduced by varying this first compound. The HABA function was thenobtained by diazotization of the phenyl residues directly on theSepharose support (Vetter et al, 1994).

(i) Sepharose-tyrosine or 2-hydroxyphenylpropionyldiamino Hexane

[0138] The primary amines tyrosine or2-hydroxyphenylpropionyl-diaminohexane dissolved in aqueous buffer (35mM borate buffer, pH 8.5) were added to the p-nitrophenylcarbonate-activated Sepharose (carrying 50-100 mmoles of active group/gof wet gel).

[0139] Reactions were carried out on 3-5 g of gel, in a total volume of12-15 ml and using a molar ratio of 3:1 between the primary amine andthe activated groups of the gel. Suspensions were gently stirred for 150minutes at room temperature, and the gels were then washed with water,MeOH, EtOAc and then, MeOH and water again. Unreacted active groups inthe gel were hydrolyzed by 5 minutes exposure to 0.2M NaOH. Gels werewashed again and resuspended in 0.2M KOH (3 g/5 ml) for the finaldiazotization step.

(ii) Diazotization Reaction

[0140] Anthranilic acid and NaNO₂ were dissolved in H₂O (156 mmoles/mlfor both) and concentrated HCl (100 ml/ml of water) was added aftercooling in an ice bath. The solution was stirred for 5 minutes and thenadded dropwise to the gel suspended in 0.2M KOH (3 g/5 ml). The reactionwas gently stirred for 15 minutes, while the temperature was controlledusing an ice bath, and the pH was monitored constantly and adjusted to8.0-8.5 using diluted KOH. A molar ratio of 1:1 between anthranilic acidand the phenyl residues in the gel was used, assuming that a completeconversion of the activated p-nitrophenyl groups occurred in theprevious step of the synthesis. The GEL A obtained was then washed (sameprocedure as in previous step) and suspended in PBS.

5.3.b Preparation of GEL B

[0141] Hydroxyl groups of Sepharose CL-4B were first activated withN,N′-disuccinimidyl-carbonate (Wilchek and Miron, 1985), and theactivated gel was then coupled to tyramine via the amino group. The HABAderivative was obtained by diazotization of the phenyl residues usinganthranilic acid

(i) Sepharose-tyramine

[0142] Tyramine dissolved in PBS (pH 7.4) was added to theN,N′-disuccinimidylcarbonate activated Sepharose (carrying 20-80 mmolesof active groups/g of wet gel). Reactions were carried out on 3-5 g ofgel, in a total volume of 12-15 ml and using a molar ratio of 5:1between the primary amine and the activated groups of the gel.Suspensions were gently stirred overnight at 4° C. Gels were then washedextensively until no more free amine could be detected and unreactedactive groups in the gel were hydrolyzed by 5 minutes exposure to 0.2MNaOH. Gels were washed again and resuspended in 0.2M borate buffer (pH8.5) (3 g/5 ml) for the final diazotization step.

(ii) Diazotization Reaction.

[0143] Anthranilic acid and NaNO2 were dissolved in H₂O and concentrated0.2M HCl was added after cooling in a ice bath. The solution was stirredfor 5 minutes and then added dropwise to the gel suspended in 0.2Mborate buffer (3 g/5 ml). The reaction was gently stirred for 15minutes, while the temperature was controlled using an ice bath and thepH monitored constantly and adjusted to 8.0-8.5 using diluted KOH. Amolar ratio of 1:1 between anthranilic acid and the phenyl residues inthe gel was used, assuming that a complete conversion of the activatedp-nitrophenyl groups occurred in the previous step of the synthesis. TheGEL B obtained was then washed and finally suspended in PBS.

5.3.c Affinity Purification of Anti-HABA Polyclonal Antibodies with GELA and GEL B

[0144] Sepharose GELS A and B were pre-treated with 0.1M TEA pH 11.5before any further use and re-equilibrated with PBS. Rabbits' antiseradiluted 1:1 with PBS or IgG antibodies obtained by (NH₄)₂SO₄precipitation were incubated with the gel for 4 hours at 4° C. Totalremoval of anti-HABA antibodies from supernatant was verified by dotblot on nitrocellulose paper, using BSA-HABA for dotting. In order toobtain an efficient retention of anti-HABA antibodies, a ratio of 1:1w/w between serum and gel was used. The gel was then washed extensivelywith 0.05 M Tris HCl, 0.5M NaCl, pH 7.5. Bound antibodies were finallyeluted by basic treatment, using 0.1M TEA, pH 11.5, immediatelyneutralized and dialyzed against PBS.

5.3.d Characterization of Anti-Sera and Affinity Purified Anti-HABAPolyclonal Antibodies: Screening for Anti-HABA Antibodies with DifferentSpecificities.

[0145] Purity of the affinity purified anti-HABA polyclonal antibodiesaccording to Example 5.3.c above was verified by SDS gelelectrophoresis. Concentration of an affinity purified anti-HABAantibody solution was determined spectrophotometrically using theaverage ε^(%) _(280 nm) value of 14.5 for IgGs. Specificity of anti-seraand affinity purified antibodies for different epitopes in the HABAmolecule was verified by ELISA and UV spectrophotometry.

5.3.e ELISA Assay

[0146] Ninety-six well microtiter plates (Nunc F96, Maxisorp) werecoated by overnight incubation at 4° C. with 50 μl/well of HABAylatedavidin solution (10 μg/ml in 0.05 M Na carbonate, pH 9.5). Plates werewashed three times with PBS/Tween 0.05% (PBS/T) and blocked by adding200 μl of PBS/T containing 3% BSA or 0.1% of gelatine. After 2 hoursincubation at 37° C., plates were washed three times with PBS/T.

[0147] Serial dilutions of antisera or affinity purified anti-HABAantibodies (50 μl) were then incubated for 2 h at

[0148] 37° C. When HABAylated avidin was used for the coating, theexperiment was run in duplicate and the antibodies were incubated withand without biotin in the diluting buffer. Plates were washed 3 timeswith PBS/T and incubated for 2 hours at 37° C. with 50 μl/well of asolution containing HRP-conjugated anti-rabbit antibodies (diluted1:2,500). After extensive washing with PBS/T, 100 μl ofo-phenylenediamine solution were added, the reaction stopped using 1MH₂SO₄ and the OD at 490 nm was measured after 5 minutes.

[0149] Absence of cross reactivity against the anthranilic part of theHABA molecule was verified running a control ELISA assay withBSA-anthranilic acid in the first coating. As shown in FIG. 1, anti-HABAantibodies purified on GEL A were able to recognize HABA as part of theHABAylated avidin in the absence (Γ) as well as in the presence ofbiotin (υ). However, in the absence of biotin, the anti-HABA antibodypurified on GEL B failed to recognize the HABA buried in the bindingsite. Upon addition of biotin, however, the HABA moiety was expelled andstrong binding of the anti-HABA antibody was detected (). This effectclearly depends on the procedure used for purification of the anti-HABAantibodies.

5.3. f Spectrophotometry

[0150] UV spectra of HABA (compound 0) in PBS was recorded in thepresence of the anti-HABA antibodies affinity purified in both GELs Aand B. The results indicate that:

[0151] Antibodies purified with GEL A (A-anti-HABAs) recognize the HABAmoiety when it is either in the azo or the quinone conformation.Antibodies purified with GEL B (B-anti-HABAs) can bind to the HABAmoiety only when it is in the azo conformation whereas they fail torecognize it in the quinone conformation. In this case, recognition ofthe HABAylated avidin occurs only after biotin expels HABA from thebinding pocket.

5.4 Preparation of Anti-HABA Monoclonal Antibodies 5.4.a HybridomaProduction

[0152] A HABA-KLH conjugate prepared as described above and carrying˜100 molecules of HABA/protein was used for the immunization. FiveBalb/c mice were first injected into the foot pads with a purepreparation of the HABA-KLH (2.5 mg/mouse, emulsified in completeFreund's adjuvant) and two weeks later subcutaneously in incompleteFreund's adjuvant. Two aditional injections were given at two weeksinterval, subcutaneously, in PBS. The mouse chosen for fusion receivedan intraperitoneal injection of the immunogen, followed the day after byan intravenous one. Three days after the i.v. injection, lymphocitesfrom the spleen and the inguinal lymph nodes (192×10⁶ cells) were fusedwith 50×10⁶ NSO/1 myeloma variant (NSO cells, kindly provided by C.Milstein, MRC, Cambridge, U.K.) by standard techniques. The fused cells(hybridomas) were distributed into 16-microculture plates (˜3×10⁴cells/well). Hybridomas that were found to secrete anti-HABA antibodieswere cloned and recloned by the limiting dilution technique. Theimmortalyzed hybridomas selected after the screening were frozen andused later for a large scale preparation of pure anti-HABA monoclonalantibodies.

5.4.b Screening Methods for Hybridoma Selection

[0153] In order to isolate anti-HABA hybridomas with desiredcharacteristics, namely (i) high affinity and specificity for the ‘HABAcore’ of the antigen, thus reducing the influence of the spacer arm inthe recognition process, and (ii) high affinity and specificty for thewhole Compound 17 (HABA+spacer arm), for isolation of an antibody thatrecognizes the molecule only when the spacer arm is present, twowell-known screening techniques were used: ‘direct’ and competitiveELISA.

[0154] For preliminary screening, a direct binding assay as describedfor polyclonal antibodies testing in 5.2.c was used, except that singlehybridoma culture supernatant was used for the serial dilutions insteadof anti-sera, and HRP-conjugated anti-mouse antibodies(Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L)diluted1:5,000) were used for detection.

[0155] The hybridomas that showed the best response in this generalELISA test were selected for further screening by competitive ELISAassay: 96-well microtiter plates (Nunc F96 Maxisorp) were coated byovernight incubation at 4° C. with 50 μl/well of a BSA-HABA solution 10μg/ml in 0.05 M NaHCO₃, pH 9.3. Plates were washed three times withPBS/Tween 0.05% (PBS/T) and blocked by adding 200 μl of PBS/T containing3% BSA. After 2 hours incubation at 37° C., plates were washed threetimes with PBS/T.

[0156] The BSA-HABA coated plates were then incubated for 2 h at 37° C.with diluted hyridoma supernatant together with different amounts ofthree HABA-derivatives (0.1-1 mg in a final volume of 50 μl): Compound 0(HABA), Compound 16 with A=(CH₂)₂, and analog of Compound 16 withA=CH═CH. The hybridoma dilutions were chosen according to the results ofthe preliminary direct binding assay in order to have approximately thesame response in the ELISA for all the hybridoma tested andcorresponding to 50% of the maximal response.

[0157] Plates were then washed 3 times with PBS/T and incubated for 2hours at 37° C. with 50 μl/well of a solution containing HRP-conjugatedanti-mouse antibodies (1:5,000). After extensive washing with PBS/T, 100μl of ABTS solution were added and the O.D. at 405 nm was measured after30 minutes.

[0158] Hybridomas giving the desired results, e.g. 745.7, 913.1 and915.65 were afterwards selected for further preparation of supernatent.

5.4.c Labeling of Anti-HABA Antibodies

[0159] Different labels can be introduced in the HABA-binding molecules,i.e. avidin and anti-HABA antibodies, for the visualization of theHABA/anti-HABA complexes. Color enzymes, colored, fluorescent andchemiluminescent probes can be covalently bound by using generallabeling protocols as described elsewhere (Garman, 1997).

Example 6 DNA and RNA Technology 6.a HABAylated Nucleotides

[0160] HABAylated nucleotides, which are suitable substrates for DNA andRNA polymerases in vitro, can be synthesized from the amino reactiveHABA-Su derivatives, such as for example Compounds 17, 21 and 28, andthe aminoallyl derivatives of the nucleotides, in a similar way asdescribed for biotin-nucleotides (Langer, 1981; Shimkus, 1986).

6.b Labeling of Oligonucleotides with HABA

[0161] The HABA derivatives that are reactive towards primary aminogroups, such as for example Compounds 17, 21 and 28,can be used forchemical labeling of amino functionalized short oligonucleotides asdescribed for biotin and DIG labels (Garman, 1997). The HABA probes canbe introduced at the 3′ or 5′ end or in the middle region of theoligonucleotide, depending on the strategy used for the introduction ofthe amino functions during the oligo synthesis. Alternatively, the thiolreactive HABA-derivative (Compound 18) can be used in thiolfunctionalized oligonucleotides.

6.c Labeling of Long Nucleic Acid Probes

[0162] DNA and RNA labeling can be achieved either enzymatically orchemically. Similar strategies and methods as those already in use fortagging nucleic acid with biotin or DIG can be used (Kessler, 1995;Garman, 1997).

[0163] PCR, nick translation and random primer methods can be appliedfor DNA labeling using HABAylated nucleotides. RNA polymerase can beused for RNA molecules.

Example 7 Applications of the HABA/Anti-HABA Technology

[0164] The HABA core of the HABA-derivatives of the invention can berecognized by both avidin and specific anti-HABA antibodies, leading toa color change from yellow to red in the chromophore. Such interactionsoccur also when the HABA-derivatives are covalently bound to any surfaceor protein. Thus, if the HABA compound is covalently bound to a proteinor to a DNA or RNA that is used as a ‘binder’ in a specific bioassay orbioapplication, the interaction with the target can be monitored withthe use of either avidin or anti-HABA antibodies.

[0165] Visualization and quantification of the HABA/avidin orHABA/anti-HABA complex can be carried out in two different ways: (i)preferably, using avidin or anti-HABA antibodies labeled by well-knownmethods of non-radioactive and radioactive labeling such asfluorescence, chemiluminescence, enzymatic label, etc., by the sameprocedures used in the avidin/biotin or the DIG/anti-DIGtechnologies.The choice of these labels will depend on the need of the users and onthe technology chosen (Garman, 1997); or (ii) by measuring the HABAcolor change that occurs after recognition with a regular UV-Visspectrophotometer that can detect the complexed HABA molecule insolution up to micromolar concentrations, or with more sophisticatedtechnologies that can be used for quantification of even lowerconcentrations.

[0166] The interaction of avidin with HABA is weaker than theavidin/biotin interaction. After the HABA/avidin complex is formed, theavidin molecule can be displaced by addition of a biotin solution andthe HABAylated probe can be monitored with the second independentmethod, by using the anti-HABA antibody system.

[0167] For the visualization of the target/binder complex, in a typicalexperiment, the HABAylated binder II such as an antibody, lectin, DNA orRNA, is added to a preparation such as a cell preparation, a DNA orprotein blot, containing the target molecule I, e.g. the antigen, thecarbohydrate and so on. After the complex HABAylated binderII/targetmolecule I is formed, the excess of HABAylated probe II is removed andavidin or anti-HABA antibodies (labeled or unlabeled) are applied.Visualization will depend on the application and the type of label used.

[0168] When nonlabeled avidin or anti-HABA antibodies are used, the HABAcolor change can be used for complex visualization, but this detectionsystem is not very sensitive (ε_(500 nm) of bound HABA is about 30,000).Labeled anti-HABA antibodies or avidin, for example with fluorescent,chemiluminescent, radioactive or color enzymatic labels, allow a highersensitivity. In this case, detection of the target molecule will dependon the type of label used.

[0169] If avidin (labeled or not labeled) is used for detection, aftervisualization a biotin solution can be applied to remove the coloredprobe, and then anti-HABA antibodies (labeled or not labeled) can beapplied for a second visualization. Parallel analysis with the twodetection systems can also be carried out if enough sample is available.

[0170] If non-labeled antibody or avidin is used, a labeled secondantibody or labeled anti-avidin antibody can be added for visualization.

[0171] Since the interaction with anti-HABA antibodies is stronger thanthe one with avidin, the anti-HABA technology is recommended when highsensitivity is needed.

[0172] The anti-HABA technology can be applied to any of the analyticaltechniques presently in use in biochemistry, molecular and cell biologyin a similar way as biotin and DIG are used for radioactive ornon-radioactive labeling.

[0173] In molecular and cell biology, hybridization with HABAylated RNAor DNA probes can be carried out for ‘in situ’ studies or for blotanalysis. Application of these techniques is very wide, includingdiagnosis (viruses, bacteria, genetic diseases), chromosome mapping,gene localization, gene expression, cell apoptosis detection.

[0174] Other analytical tools that use radioactive or non-radioactivelabeling and that can take advantage of the HABA technology areglycoprotein sugar analysis and classical immunoassays (e.g. ELISA).HABAylated molecules can also be immobilized to solid supports foraffinity chromatography or phage display screening as in theavidin/biotin technology.

[0175] In molecular and cell biology, this technique can be used, forexample, in blotting:

[0176] Southern blot: In order to identify the presence of a specificgene in a DNA sample, a HABAylated probe ca be used: the DNA ispreviously digested with specific restriction enzyme and is subjected toagarose electrophoresis. DNA is then transferred to a specific membrane(nylon, nitrocellulose or activated paper) and hybridized with theHABAylated complementary DNA. The anti-HABA detection system asdescribed above can be used to visualize the desired band.

[0177] Northern blot: Hybridization with HABAylated probes can becarried out in a similar way as for Southern blot. RNA molecules, e.g.mRNAs, are separated by agarose electrophoresis, transferred to aspecific membrane and hybridized with the HABAylated DNA or RNA probes.Hybridized molecules can be visualized using the anti-HABA technologypreviously described.

[0178] A further application of this technology is in chromosomemapping: HABAylated ssDNA or RNA probes complementary to the targetgenes can be used for hybridization to metaphase chromosome spreads.After washing off the excess of HABAylated probes, fluorescent orchemiluminescent anti-HABA antibodies are added and, after washing, thelocus of hybridization can be localized by light or fluorescentmicroscopy. This technique allows to visualize the location of specificgenes within, or inside, specific chromosomes.

[0179] A still further application of this technology is in mRNAexpression in specific tissues in situ hybridization hystochemistry.Cellular localization of mRNAs can be visualized using specificHABAylated riboprobes: the HABAylated RNA complementary to the targetmRNA can be hybridized with the tissue under examination. Hybridizationcan be visualized with the anti-HABA system using light or fluorescentmicroscopy. This methodology is used for localization or expressionstudies of specific mRNAs, either in different tissues or in differenttypes of cells within a single tissue.

[0180] Yet a further application of this technology is in thevisualization of apoptotic cells. Terminal transferase-mediatedd-UTP-HABA nick end labeling (TUNEL) can be used to 3′-end-labelfragmented DNA in order to visualize apoptotic cells which arecharacterized by a highly segmented DNA. This DNA can be 3′ end labeledwith a HABAylated nucleotide, and the HABAylated ends are detected bythe anti-HABA system. Visualization is carried out using light orfluorescent microscopy whereby the apoptotic cells result very intenselymarked as compared to normal ones.

[0181] In lectin cytochemistry, lectin conjugates with HABA and labeledanti-HABA/avidin are used for light or electron microscopy detection ofglycoconjugates in tissue sections. The HABAylated lectins are appliedto the tissue section and labeled anti-HABA/avidin are applied. Theinteraction can be visualized either by electron or light microscopy.

[0182] A further application of the technology of the present inventionis in diagnosis, for example in genome incorporated viruses (DNA orRNA), slow replicating bacteria and genetic diseases.

[0183] a. Genome Incorporated Viruses (DNA or RNA): In situhybridization of DNA or RNA of the target virus pathogen with specificHABAylated probes can be carried out inside infected cells. The cellsare incubated with the specific HABAylated DNA or RNA probe and, afterwashing, hybridization can be visualized after labeled avidin/anti-HABAapplication. Visualization can be achieved using a light or electronmicroscope.

[0184] b. Slow replicating bacteria: Hybridization techniques can alsobe useful for the identification of certain bacteria from patients'specimen such as slow replicating bacteria, e.g. MycobacteriumTubercolosis, or bacteria difficult to identify with common kits.Bacteria DNA can be identified by hybridization with specific HABAylatedDNA or RNA probes. The patient specimen is first treated in order toallow contact between probe and target, e.g. cell treatment withproteinase K, SDS, etc. Visualization can be carried out with amicroscope after hybridization with the avidin/anti-HABA system andwashing off the unbound probes.

[0185] c. Genetic diseases: The same principles as those described forchromosome mapping can be used for diagnosis of genetic-related diseasesand prenatal diagnosis. HABAylated DNA probes complementary to specificgenes or gene sequences are hybridized to the chromosome spread.Hybridization is visualized after anti-HABA system application, usingfluorescent or chemiluminescent labeling and microscopic visualization.

[0186] In biochemistry, the technology of the present invention can beused in Western blotting, ELISA assays, and glycoprotein sugar analysis.

[0187] For Western blotting, SDS-PAGE is carried out in the proteinmixture and Western immunoblot is carried out to transfer the proteinsto a specific membrane. After blocking the uncoated sites, HABAylatedantibodies can be introduced. Detection is carried out as describedabove using labeled avidin or anti-HABA antibodies.

[0188] For ELISA assay, a biological solution, e.g. cell extract,containing the target molecule, is used to coat the ELISA plate. Afterblocking the uncoated sites, HABAylated anti-target antibodies areapplied. After washing, enzyme-labeled avidin or anti-HABA antibodiesare incubated and enzyme color development is carried out forvisualization and quantification.

[0189] For glycoprotein sugar analysis, i.e. for analysis of the type ofglycosylation of a glycoprotein, a similar approach as the one describedabove for the ELISA test can be used. The purified glycoprotein isabsorbed on the microtiter plate surface and, after removing the unboundprotein and quenching of the uncoated sites, HABAylated specific lectinsare applied. After washing, the degree of lectin binding is measuredusing either labeled anti-HABA antibodies or labeled avidin.

[0190] Other application envisaged by the present invention refers todrug delivery studies whereby HABAylated oligonucleotides can be used tofollow the fate of oligonucleotides in drug delivery studies for gene oranti-sense therapy applications (Lappalainen, 1997).

References

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[0192] Engvall E., Enzyme Immunoassay ELISA and EMIT, Meth. Enzymol. 70,419 (1980).

[0193] Garman A., Non-Radioactive Labeling, Academic Press, 1997.

[0194] Gosh A. K. et al., N,N′-Disuccinimidyl Carbonate: A UsefulReagent for Alkoxycarbonylation of Amines, Tetrahedron Lett. 33, 2781(1992).

[0195] Kessler C., Methods for nonradiaoctive labeling of nucleic acidsin Non-radioactive probing, blotting, sequencing (ed. L. J. Kickca),Academic Press, 1995.

[0196] Kitagawa T. et al., Preparation and Characterization ofHetero-bifunctional Cross-linking Reagents for Protein Modification,Chem. Pharm. Bull. 29, 1130 (1981).

[0197] Langer P. R., Waldrop A. A., Ward D. C., Enzymatic synthesis ofbiotin-labeled polynucleotides: Novel nucleic acid affinity probes,Proc. Natl. Acad. Sci. USA 78, 6633 (1981).

[0198] Lappalainen K. al., Intracellular distribution of oligonucleotidedelivered by cationic liopsomes: light and electron microscopy study, J.Histochem. Cytochm. 45, 265 (1997).

[0199] Shimkus M.L, Guaglianone P., Herman T. M., Synthesis andcharacterization of biotin-labeled nucleotide analogs, DNA 5 (3) 247(1986).

[0200] Takeda K. et al., Convenient Methods for Syntheses of ActiveCarbamates, Ureas and Nitrosoureas using N,N′-disuccinimido Carbonate(DSC), Tetrahedron Lett. 24, 4569 (1983).

[0201] Takeda K., Ogura H., Studies on Heterocyclic Compounds XLIII.Insertion Reaction of Carbonyl groups using Disuccinimido carbonate(DSC), Synth. Commun. 12, 213 (1982).

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What is claimed is:
 1. Anti-HABA antibodies.
 2. Anti-HABA antibodiesaccording to claim 1, which are polyclonal antibodies.
 3. Polyclonalanti-HABA antibodies according to claim 2 which recognize the entireHABA molecule.
 4. Polyclonal anti-HABA antibodies according to claim 2which are specific for the azophenyl-2-carboxylic acid moiety of theHABA core.
 5. Anti-HABA antibodies according to claim 1, which aremonoclonal antibodies.
 6. A method for qualitative or quantitativedetection of a molecule I in a sample which comprises the steps of: a.contacting the sample with a HABAylated molecule II that recognizesmolecule I; b. reacting with labeled anti-HABA antibodies or labeledavidin; c. visualizing and quantifying the HABA/avidin or HABA/anti-HABAcomplex formed.
 7. The method according to claim 6 for the detection ofa DNA molecule transferred to a specific membrane after digestion of aDNA sample with specific restriction enzymes and separation by agaroseelectrophoresis, which comprises hybridizing the DNA molecule with theHABAylated DNA probe.
 8. The method according to claim 6 for detectingan RNA molecule transferred to a specific membrane after separation ofan RNA sample by agarose electrophoresis, which comprises hybridizingsaid RNA molecule with the hybridized DNA or RNA probe.
 9. The methodaccording to claim 6 for the detection of a protein molecule transferredto a specific membrane after separation of a protein mixture bySDS-PAGE, which comprises reacting said protein molecule with thecorresponding HABAylated antibody.